proNGF inhibits proliferation and oligodendrogenesis of ... - Core

Stem Cell Research (2013) 11, 874–887

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proNGF inhibits proliferation and oligodendrogenesis of postnatal hippocampal neural stem/progenitor cells through p75NTR in vitro Jingjing Guo a , Jianing Wang a , Chunrong Liang b , Jun Yan a , Yeran Wang b , Gaoxiang Liu a , Zhenzhou Jiang a , Luyong Zhang a , Xiaobin Wang c , Yanjiang Wang b , Xinfu Zhou d , Hong Liao a,e,⁎ a

Neurobiology Laboratory, Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, China Department of Neurology and Center for Clinical Neuroscience, Daping Hospital, Third Military Medical University, Chongqing 400042, China c Laboratory Animal Center, Southeast University, Nanjing 210009, China d School of Pharmacy and Medical Sciences, Sansom Institute, University of South Australia, Adelaide, SA 5000, Australia e State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China b

Received 26 September 2012; received in revised form 2 May 2013; accepted 7 May 2013 Available online 21 May 2013

Abstract Neural stem/progenitor cells (NSCs) proliferate and differentiate under tight regulation by various factors in the stem cell niche. Recent studies have shown that the precursor of nerve growth factor (NGF), proNGF, abounds in the central nervous system (CNS) and that its expression level in the brain is substantially elevated with aging as well as in several types of CNS disorders. In this study, we found for the first time that proNGF inhibited the proliferation of NSCs isolated from postnatal mouse hippocampus and caused cell cycle arrest in the G0/G1 phase without affecting apoptosis. In addition, proNGF reduced the differentiation of NSCs to oligodendrocytes. The effects of proNGF were blocked by the fusion protein of p75 neurotrophin receptor extracellular domain and human IgG Fc fragment (p75NTR/Fc), and by p75NTR knockout, suggesting that proNGF/ p75NTR interaction was involved in the effects of proNGF on NSC proliferation and differentiation. proNGF decreased the phosphorylation level of extracellular signal responsive kinase 1/2 (ERK 1/2) in a p75NTR-dependent manner under both self-renewal and differentiation conditions. The inhibition of ERK 1/2 phosphorylation by U0126 significantly reduced the proliferation and oligodendrogenesis of NSCs, indicating that ERK 1/2 inhibition by proNGF partially explains its effects on NSC proliferation and oligodendrogenesis. These results suggest that the proNGF/p75NTR signal plays a key role in the regulation of NSCs' behavior. © 2013 Elsevier B.V. All rights reserved. Abbreviations: proNGF, the precursor of nerve growth factor; p75NTR, p75 neurotrophin receptor; NSCs, neural stem/progenitor cells; BrdU, 5-bromo-2-deoxyuridine; TUNEL, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling; ERK, extracellular signal responsive kinase. ⁎ Corresponding author at: Neurobiology Laboratory, Jiangsu Center for Drug Screening, China Pharmaceutical University, 24# Tongjiaxiang, Nanjing 210009, China. Fax: + 86 25 83271142. E-mail address: [email protected] (H. Liao). 1873-5061/$ - see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scr.2013.05.004

Effects of proNGF on hippocampal NSCs via p75NTR

Introduction Neural stem/progenitor cells (NSCs) proliferate and differentiate throughout life to maintain the constant size of the NSC pool and to produce newly born cells for reparation of CNS injury. These processes are tightly regulated by various factors from the microenvironment/niche where NSCs reside (Wurmser et al., 2004). The elucidation of potential signaling molecules regulating NSC activity in the niche may contribute not only to the understanding of neurogenesis but also to the development of new therapies for nervous system disorders. The unprocessed precursor of nerve growth factor (NGF), proNGF, has been demonstrated to actually be the predominant form of NGF in CNS (Fahnestock et al., 2001). It has emerged from studies in the past decade that proNGF has distinct functions with respect to NGF, besides acting chaperone-like for NGF folding during its biogenesis. Unlike NGF, which is well-known for its beneficial effects on cell survival, proliferation, and neurite outgrowth (Cattaneo and McKay, 1990; Wang et al., 2009), proNGF has been mostly shown to induce apoptosis of various types of cells under pathological conditions including seizures and spinal cord injury, where its expression is elevated (Volosin et al., 2008; Beattie et al., 2002). Furthermore, an elevated proNGF level was observed in the brains of old rodents and patients with Alzheimer's disease (Al-Shawi et al., 2008; Pedraza et al., 2005; Terry et al., 2011). proNGF was postulated to be accumulated in the hippocampus which contains a population of NSCs with a decreased ability to generate new cells. These studies prompted us to investigate whether proNGF constitutes a potential factor that regulates the activity of hippocampal NSCs. The p75 neurotrophin receptor (p75NTR) has been identified as the common receptor for neurotrophins and proneurotrophins. As it lacks an intrinsic ligand-inducible enzymatic activity, p75NTR functions through either the collaboration with co-receptors, the recruitment of intracellular signaling proteins or the release of its intracellular domain via secretasedependent proteolysis (Hempstead, 2002). p75NTR is involved in multiple cellular responses including cell apoptosis, survival, neurite outgrowth, migration, and cell cycle arrest through the activation of various downstream signaling pathways (Hempstead, 2002; Cragnolini and Friedman, 2008). Recent studies have identified the expression of p75NTR in NSCs from the subventricular zone and progenitor cells in dentate gyrus, and have proposed that p75NTR deficiency perturbed NSC proliferation and differentiation in response to neurotrophins (Bernabeu and Longo, 2010; Young et al., 2007). These observations suggested that p75NTR signaling might be involved in regulating the proliferation and differentiation of NSCs. Moreover, studies have already confirmed that proNGF, among the neurotrophin family, preferentially binds p75NTR with high affinity and initiates apoptosis in several neuronal subpopulations (Lee et al., 2001). Our previous studies suggest that proNGF retards neurite outgrowth in both neuronal cell lines and primary neuron through p75NTR (Wang et al., 2010). Furthermore, it is well established that p75NTR is substantially increased under pathological conditions that are known to induce proNGF expression (Chakravarthy et al., 2012; Podlesniy et al., 2006). Therefore, it is reasonable to investigate whether proNGF/ p75NTR interaction plays a role in regulating the behaviors of NSCs.

875 In this study, the results showed that proNGF inhibited NSC proliferation and reduced their differentiation to oligodendrocyte in a p75NTR-dependent manner, which involved inhibition of the ERK 1/2 signaling pathway.

Materials and methods Cell culture All animal tests were carried out in accordance with the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals published by the US National Academy of Sciences (http://oacu.od.nih.gov/regs/index.htm). All experimental procedures were approved by the Administration Committee of Experimental Animals, Jiangsu Province and China Pharmaceutical University. NSCs were isolated from the hippocampus of newborn pups (postnatal day 1, C57BL/6J strain) of wild type and p75NTR knock-out mice (p75NTRExonIII−/− mice, Jackson lab, West Grove, PA, USA) following previously described methods with some modifications (Shetty and Turner, 1998). In brief, whole hippocampi were dissected, dissociated in Accutase Cell Dissociation Reagent (Invitrogen, Carlsbad, CA, USA) and seeded in the proliferation medium consisting of DMEM/F12, B27 (2%, Invitrogen), bFGF (20 ng/ml, Invitrogen), EGF (20 ng/ml, Invitrogen) and 1% penicillin–streptomycin. Cells proliferated and formed neurospheres after 3–4 days of culture. After one or two passages, cells were used in the subsequent experiments. In the assays for multipotency, single cells dissociated from neurospheres were plated in 24-well plates with coverslips pre-coated with poly-D-lysine (PDL, Sigma-Aldrich, St Louis, MO, USA) and laminin (Invitrogen), and were cultured in DMEM/ F12 containing 2% B27 and 1% FBS to help the differentiation of cells. Primary rat hippocampal neurons were cultured following methods described previously (Volosin et al., 2008). Cells isolated from the hippocampus of rat fetuses (embryonic day 18, E18) were plated on poly-L-lysine (PLL, Sigma)-coated 24-well plates and maintained in Neurobasal™ medium (Invitrogen) supplemented with 2% B27 and Glutamax (1%, Invitrogen) for four days before treatment.

Cell viability assay Single dissociated NSCs were plated in replicates of four in 96-well plates at a density of 7000 cells per well in 100 μl DMEM/F12 containing 2% B27, 10 ng/ml EGF and 10 ng/ml bFGF. Cells were treated with proNGF (0.4–2 nM in PBS, cleavage-resistant mutated form, human; Alomone, Israel) for 24 h or 48 h with PBS as a control, or U0126 (1 and 10 μM, in 0.2% DMSO; Beyotime, Jiangsu, China) for 48 h with DMSO (0.2%) as a control. Cell proliferation was quantified using a CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol.

BrdU incorporation assay Single dissociated cells were plated in 24-well plates with PLL-coated coverslips and cultured with DMEM/F12 containing 2% B27, 10 ng/ml EGF and 10 ng/ml bFGF.

876 5-Bromo-2-deoxyuridine (BrdU, 10 μg/ml; Sigma) was added to the cultures 36 h before fixation. Nuclei that incorporated BrdU in this time-window were detected by immunofluorescence as described below. The cell proliferation rate was represented by the percentage of BrdU-positive cells (total number of BrdU-positive cells / total cell number evaluated by Hoechst staining).

Neurosphere assay Dissociated NSCs were plated at a density of 100,000 cells/ml in uncoated 35-mm dishes. Cells were cultured in proliferation medium with or without 2 nM proNGF for 24 h and 48 h. At the end of the culture, five random viewfields of each group were captured using an Olympus microscope (New Hyde Park, NY, USA, http://www.olympus.com/), and the diameters of neurospheres were measured using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The assay was conducted in triplicate.

TUNEL assay Cells were plated on PLL-coated coverslips and were treated with PBS control or 2 nM proNGF for 48 h. Cells were then processed for TUNEL analysis (Roche Molecular Biochemicals, Indianapolis, IN, USA) and counterstained with Hoechst 33342 to visualize nuclei. TUNEL-positive cells of at least 10 random viewfields containing more than 2000 cells were counted for each culture condition.

Cell cycle assay Cells in G1/G0, S, and G2/M phases of the cell cycle can be discriminated by analysis of DNA ploidy (Darzynkiewicz and Juan, 2001; Nunez, 2001). Here, the measurement of cellular DNA content and the analysis of cell cycle were performed using propidium iodide (PI) staining and flow cytometry. Briefly, NSCs were dissociated and fixed in ice-cold 70% ethanol. After washing with PBS, cells were resuspended in PBS containing 50 μg/ml PI (Sigma) and 10 μg/ml RNase A (Beyotime). Cell suspensions were then incubated at 37 °C for 30 min and then transferred to FACS Calibur flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA, http://www. bdbiosciences.com/). PI fluorescence was measured and the percentage of cells existing within various phases in the cell cycle was analyzed using ModFit software (http://www.vsh. com/products/mflt/index.asp). Data from a minimum of 10,000 cells per sample were collected.

Reverse-transcription PCR and real-time PCR Total mRNA of NSCs was isolated using TRIzol Reagent (Invitrogen). RNA was reverse-transcribed using the RevertAid™ First Strand cDNA Synthesis Kit (Fermantas, Amherst, NY, USA) with oligodT. PCR was performed with GoTaq Flexi DNA Polymerase (Promega). Amplified PCR products were analyzed by electrophoresis on a 1% agarose gel. For real-time PCR, an abundance of each mRNA sample was quantified using the corresponding primers and the SYBR® Premix Ex Taq™II (Takara, Japan). Gene expressions

J. Guo et al. were normalized by β-actin and represented as a relative transcription level to those in the PBS-treated group. PCR primer sets were shown in Supplementary Table S1.

Western blot Protein samples from cells and tissues were extracted using RIPA buffer (Beyotime) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Equal amounts of extracts from different preparations were separated on SDS-polyacrylamide gels and subjected to immunoblotting. Primary antibodies against the extracellular domain of p75NTR (rabbit, 1:1000, a gift from Professor Moses Chao of New York University), the cytoplasmic domain of p75NTR (rabbit, 1:700, Promega), sortilin (rabbit, 1:1000, Abcam), TrkA (rabbit, 1:1000, Abcam), caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (rabbit, 1:1500, Cell Signaling Technology), cyclin E (rabbit, 1:800, Cell Signaling Technology), p-ERK1/2 (rabbit, 1:1000, Cell Signaling Technology), total ERK1/2 (rabbit, 1:1000, Cell Signaling Technology), p-Akt (rabbit, 1:1000, Cell Signaling Technology), Akt (rabbit, 1:1000, Cell Signaling Technology), p-STAT3 (rabbit, 1:1500, Cell Signaling Technology), STAT3 (rabbit, 1:1500, Cell Signaling Technology), β-tubulin III (mouse, 1:800, Chemicon, Temecula, CA, US), GFAP (rabbit, 1:2000, DAKO, Copenhagen, Denmark), CNPase (mouse, 1: 1000, Sigma), NG2 (rabbit, 1:600, Chemicon), nestin (mouse, 1:500, Chemicon) and β-actin (mouse, 1:3000, Sigma) were used. To detect the phosphorylated protein and total protein of ERK 1/2, Akt and STAT3, blots were probed with antibodies against the phosphorylated protein, stripped and then reprobed for total protein. Immunoreactive bands were visualized by incubation with Horseradish peroxidase (HRP)-conjugated secondary antibodies and detection using ECL (Pierce, Rockford, IL, USA). Relative expression levels of proteins were quantified using Quantity One software (Bio-Rad).

Immunocytochemistry Cells on coverslips were processed for immunocytochemistry as previously described (Liao et al., 2008). Primary antibodies against BrdU (mouse, 1:200, Santa Cruz Biotechnology, CA, USA), nestin (mouse, 1:200, Chemicon), β-tubulin III (mouse, 1:300, Chemicon), GFAP (rabbit, 1:500, DAKO), CNPase (mouse, 1: 800, Sigma), NG2 (rabbit, 1:300, Chemicon), p75NTR intracellular domain (rabbit, 1:700, Promega) and p75NTR extracellular domain (rabbit, 1:1000, a gift from Professor Mose Chao of New York University) were used. Then samples were incubated with Alexa Fluor 488 and/or Cy3-conjugated secondary antibodies (1:300, Invitrogen), counterstained with Hoechst 33342 to label all cell nuclei and mounted in fluorescent mounting medium. For the BrdU assay, cells were incubated in 2 N HCl for 30 min at room temperature before blocking. The fluorescence imaging was visualized by using an Olympus fluorescence microscope (New Hyde Park, NY, USA, http://www.olympus.com/). At least 2000 cells from 10 to 15 viewing fields per group were used to calculate the percentage of cells.


Statistical analysis Statistical analysis was carried out with Graph Pad Prism 5 software (Graph Pad Software Inc., San Diego, CA). Data were presented as a mean ± SEM of at least three independent experiments. For experiments with more than two groups, results were compared using one-way analysis of variance (ANOVA) with post hoc Tukey's test. For two group-designed experiments, comparisons were performed using unpaired Student's t-test. A p-value of b 0.05 was considered statistically significant.

Results proNGF inhibited NSC proliferation without inducing any apoptosis Dissociated NSCs were maintained in proliferation medium during 24 or 48 hour blocks with or without proNGF (0.4 nM, 2 nM, 10 nM, respectively). The results of cell viability assay demonstrated that proNGF induced a significant reduction in viable cell number (Fig. 1A), which may have resulted from a decrease in the proliferation and/or induction of cell apoptosis. To investigate whether proNGF inhibited NSC proliferation, BrdU incorporation assay and neurosphere assay were performed. As shown in Figs. 1B and C, the percentage of BrdU-labeled cells was decreased by about one fifth in the

877 presence of 2 nM proNGF for 48 h (p b 0.001), and the neurosphere size was significantly reduced by proNGF treatment for 24 and 48 h blocks, suggesting that proNGF exerted an inhibitory effect on NSC proliferation. Mounting evidence has presented proNGF as a potential inducer of cell apoptosis in several types of neural cells (Hempstead, 2009). To explore the possibility that the decrease in viable cells was partially attributed to elevated cell apoptosis, we performed terminal deoxynucleotidyl transferasemediated biotinylated UTP nick end labeling (TUNEL) assay which reflects apoptosis-associated DNA strand breaks. As shown in Figs. 1D and E, no significant difference in TUNELpositive cells was observed between PBS- and proNGF-treated NSCs (p N 0.05). Furthermore, proNGF did not impact the cleavage of caspase-3 in NSCs, a critical step in inducing cell apoptosis (Fig. 1F). As it has been reported that proNGF induced apoptosis of hippocampal neurons both in vitro and in vivo (Volosin et al., 2008), we exposed hippocampal neurons derived from E18 rat fetus to proNGF (2 nM) for 48 h. The proportion of TUNEL-positive neurons was increased by around three folds after proNGF treatment (Figs. 1D and E), which was in line with previous studies. Taken together, proNGF inhibited the proliferation of NSCs without inducing apoptosis.

proNGF retarded cell cycle progression The cell cycle is involved in the regulation of cell growth. PI staining and flow cytometry were applied to monitor the

Figure 1 proNGF inhibits the proliferation of hippocampal NSCs without inducing apoptosis. (A) NSCs were treated with serially diluted proNGF for 24 h or 48 h followed by an assessment of cell viability assay. (B) proNGF (2 nM) reduced the percentage of BrdU-incorporating cells (BrdU, red; Hoechst, blue). (C) proNGF reduced the diameters of neurospheres after 24 and 48 h in culture. (D) proNGF induces apoptosis of rat embryonic hippocampal neurons but not that of NSCs, as shown by TUNEL assay (TUNEL, green; Hoechst, blue). (E) Percentages of TUNEL-positive cells among total cells were calculated. (F) proNGF did not impact the cleavage of caspase-3 in NSCs. The error bars in bar graphs represent SEM of three (C, E, F) or five (B) independent experiments. *p b 0.05, **p b 0.01, ***p b 0.001 compared with PBS group. N.S., no significant difference. Scale bar = 50 μm.

878 allocation of cells to different cell cycle phases under self-renewing conditions in the presence or absence of 2 nM of proNGF for 24 and 48 h. Compared with PBS control, proNGF treatment increased the percentage of cells in the G0/G1 phase at the expense of the S-phase (Figs. 2A and B). No statistically significant difference in the G2/M phase distribution was noted between the two groups (Fig. 2C). Cell cycle progression is tightly controlled by a series of regulators. Both cyclin D1 and cyclin E accelerate G1-to-S transition. Real-time PCR and western blot analyses revealed that proNGF treatment reduced the expression of cyclin E at both mRNA (Fig. 2G) and protein levels (Figs. 2H and I) by more than 40% and 20% respectively. However, neither mRNA nor the protein expression level of cyclin D1 was significantly affected by proNGF (Figs. 2D–F). These results imply that proNGF probably inhibits NSC proliferation by arresting cells at the G0/ G1 phase.

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proNGF inhibited NSC proliferation in a p75NTR-dependent manner p75NTR is the high-affinity receptor for proNGF, as first proposed by Ramee Lee et al. (2001). To explore whether p75NTR signaling was involved in the effect of proNGF on NSC proliferation, we first tested the expression of p75NTR in NSCs. mRNA and protein expression of p75NTR in NSCs were detected by RT-PCR and western blot respectively, with mouse brain lysate as a positive control (Figs. 3A and B). A protein band for p75NTR was absent in NSCs from p75NTR-knockout mice (Fig. 3B). The immunostaining demonstrated that p75NTR colocalized with the stem cell marker nestin in neurospheres (Fig. 3C-a), which was further confirmed on dissociated NSCs using antibodies against the extracellular (Fig. 3C-b) and the intracellular domain (Fig. 3C-c) of p75NTR. The specificity of antibodies against

Figure 2 proNGF retards the cell cycle progression of hippocampal NSCs. proNGF significantly increased the relative distribution of NSCs in G0/G1 phase (A) while reducing their distribution in S phase (B). No significant difference was found in G2/M distribution between groups (C). Real-time PCR analysis demonstrated that proNGF did not impact the expression levels of cyclin D1 (D) but it downregulated cyclin E in NSCs (G). Immunoblotting of cyclin D1 (E) and cyclin E (H) in NSCs extracts showed consistent effects of proNGF on their expression at the protein level. β-Actin was used as a loading control. Bar graphs show the relative protein levels of cyclin D1 (F) and cyclin E (I). Data were shown as means ± SEM of three independent experiments. *p b 0.05, **p b 0.01 between groups.


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Figure 3 proNGF inhibits NSC proliferation in a p75NTR-dependent manner. (A) RT-PCR analysis revealed the expression of p75NTR in NSCs, with mouse brain extracts as a positive control. (B) Protein lysates from NSCs and wild-type mouse brain were probed for the presence of p75NTR using antibody against the extracellular domain of p75NTR. Note that a protein band for p75NTR was absent in p75NTR-knockout NSCs. (C) Double immunostaining with antibodies against the extracellular domain of p75NTR (green) and nestin (red) demonstrated the expression of p75NTR on NSCs spheres (a) and dissociated cells (b). (c) Another antibody raised against the intracellular domain of p75NTR was applied to further confirm the expression of p75NTR on dissociated NSCs. Cell nuclei were all counterstained by Hoechst (blue). (D) p75NTR/Fc (140 nM) partially reversed the inhibitory effect of proNGF (2 nM) on NSC proliferation as shown by the fluorescence micrographs of BrdU immunostaining (BrdU, red; Hoechst, blue). (E) BrdU incorporation rate in wild-type and p75NTR-deficient NSCs was quantified following proNGF (2 nM) treatment for 48 h (BrdU, red; Hoechst, blue), which showed no effect of proNGF on the proliferation of p75NTR-deficient NSCs. Values in bar graphs represent the mean ± SEM from three independent experiments. *p b 0.05, **p b 0.01, ***p b 0.001 between groups. Scale bar = 50 μm. Abbreviations: WT, wild type; p75-KO, p75NTR-knockout; p75ECD, p75NTR extracellular domain; p75ICD, p75NTR intracellular domain.

nestin and p75NTR extracellular domain was confirmed by western blot as shown in Supplementary Fig. S1. We next investigated the functional interaction between proNGF and p75NTR in NSCs. The fusion molecule of p75NTR

extracellular domain and human IgG Fc fragment (p75NTR/ Fc) was used to scavenge proNGF from the culture medium. p75NTR/Fc has been shown to be efficient in antagonizing the pro-apoptotic effect of proNGF on neurons (Wang et al.,

880 2010). Cells were exposed to proNGF (2 nM) alone and together with p75NTR/Fc (140 nM) for 48 h. As shown in Fig. 3D, the addition of p75NTR/Fc largely reversed the effect of proNGF on NSC proliferation (p b 0.001, vs. proNGF group). However, p75NTR/Fc alone had no effect on the proliferation (p N 0.05, vs. PBS group). To provide further evidence for the involvement of p75NTR in proNGF-induced inhibition of cell proliferation, NSCs derived from p75NTR-knockout mice were treated with proNGF (2 nM) for 48 h. In contrast to wild-type NSCs, p75NTR-deficient NSCs displayed minimal difference in the BrdU incorporation rate after proNGF treatment (Fig. 3E). Together, these results point to a critical role of p75NTR in mediating the inhibitory effect of proNGF on NSC proliferation.

proNGF suppressed ERK1/2 phosphorylation which affected NSC proliferation under self-renewal condition Several signaling pathways including MEK/ERK 1/2, PI3K/Akt and JAK/STAT3 are involved in NSC proliferation according to previous studies (Migita et al., 2008; Kang and Kang, 2008). We next explored the possible involvement of these signaling pathways in the effect of proNGF on NSC proliferation. As shown in Fig. 4A, resting NSCs obtained by starvation in a growth factor-free medium displayed a prompt increase in ERK 1/2 phosphorylation level within 30 min, upon the stimulation by the proliferation medium containing EGF and bFGF. proNGF (2 nM) treatment transiently suppressed the phosphorylation of ERK 1/2 (p b 0.05, vs. PBS group, Figs. 4B and C). However, no significant difference was found in the phosphorylation level of Akt and STAT3 between PBS- and proNGF-treated groups (Supplementary Fig. S2). We then focused on the ERK 1/2 signaling in subsequent experiments. The inhibitory effect of proNGF on ERK 1/2 activation was partially reversed by simultaneous p75NTR/Fc (140 nM) treatment (p b 0.05, vs. proNGF group, Figs. 4D and E). Nonetheless, proNGF (2 nM) failed to decreased the phosphorylation level of ERK 1/2 in p75NTR-knockout NSCs compared with PBS control (p N 0.05, vs. PBS group, Figs. 4G and H), even though similar activation of ERK 1/2 was observed upon proliferation (Fig. 4F). To confirm the role of ERK 1/2 inhibition in NSC proliferation, NSCs were treated with U0126, a selective inhibitor of MEK 1/2, the kinase immediately upstream of ERK 1/2.

J. Guo et al. CellTiter-Glo cell viability assay and BrdU incorporation assay were performed 48 h later to assess NSC proliferation. As shown in Figs. 4I and J, various concentrations of U0126 significantly inhibited NSC proliferation. Ten micromolar of U0126 caused a remarkable decrease in the phosphorylation level of ERK 1/2 upon proliferation (Fig. 4K). Taken together, these results imply that inhibition of ERK 1/2 phosphorylation by proNGF through p75NTR under the self-renewal condition accounted, at least to some extent, for the effect of proNGF on NSC proliferation.

proNGF changed the lineage fate of NSCs To investigate the effect of proNGF on NSC differentiation, we cultured dissociated NSCs in PDL/laminin-coated 24-well plates with DMEM/F12 containing B27 and 1% FBS for seven days. As shown in Figs. 5A and B, the percentage of β-tubulin III-positive (a specific marker of neuron) cells was not affected, while a slight increase of GFAP-positive (a specific marker of astrocyte) cells was observed. Interestingly, the percentage of NG2-positive (a specific marker of immature oligodendrocyte) and CNPase-positive (a specific marker of mature oligodendrocyte) cells were decreased by about one third and one fourth respectively compared with PBS control, resulting in a significant reduction in total oligodendrocytes (the sum of NG2- and CNPase-positive cells) in the proNGF-treated group (p b 0.05, compared with the PBS group). Consistently, the expression of GFAP, β-tubulin III, CNPase and NG2 at protein level was modulated to a similar extent in NSCs in response to 2 nM proNGF treatment (Fig. 5C). Next, mRNA expression levels of several lineage-restricted genes were determined after a 24-hour or a 4-day differentiation. In addition to GFAP and nestin, Olig1, Olig2 and Ngn1 mRNAs were also evaluated, among which the former two encode proteins that are essential for oligodendrogenesis, with the latter being for neurogenesis in CNS. As shown in Fig. 5D, Olig2 mRNA levels were reduced by about 60% while GFAP mRNA was elevated by about 50% in proNGF-treated cells, compared with PBS control at 24 h after differentiation. On the fourth day of exposure to proNGF, Olig1 mRNA decreased by about 40% (Fig. 5E). However, there were no statistically significant differences in nestin and Ngn 1 mRNA levels between the two groups.

Figure 4 proNGF inhibits ERK1/2 phosphorylation through p75NTR under the self-renewal condition. (A) Representative immunoblotting showed that the phosphorylation level of ERK1/2 in NSCs varied with time after the resting cells were transferred to proliferation medium containing EGF and bFGF. (B) Quiescent NSCs were treated with or without 2 nM proNGF in proliferation medium followed by western blot to detect ERK1/2 phosphorylation levels. (C) The densitometric quantification showed that proNGF inhibited ERK 1/2 phosphorylation at 30 min. (D) Representative immunoblots showed p-ERK 1/2 and total ERK 1/2 expressions in NSCs treated with proNGF (2 nM) alone or together with p75NTR/Fc (140 nM) upon proliferation. (E) The densitometric quantification revealed that p75NTR/Fc partially reversed the effect of proNGF on ERK 1/2 activation. (F) ERK 1/2 phosphorylation levels in p75NTR-knockout NSCs at the indicated time points to proliferation. (G) proNGF did not impact ERK 1/2 phosphorylation in p75NTR-deficient NSCs, as revealed by quantitative analysis from western blot results (H). (I) U0126 (in 0.2% DMSO) at various concentrations significantly inhibited NSC proliferation as determined by CellTiter-Glo cell viability assay. 0.2% DMSO was used as a solvent control. (J) U0126 (10 μM, in 0.2% DMSO) dramatically reduced the BrdU incorporation rate of NSCs (BrdU, red; Hoechst, blue). (K) Representative immunoblots showed the inhibitory effect of U0126 (10 μM) on ERK 1/2 phosphorylation in NSCs at the indicated time points after the initiation of cell proliferation. Values in bar graphs represent the mean ± SEM from three independent experiments. *p b 0.05, **p b 0.01, ***p b 0.001 between groups (C and E) or compared with the DMSO control group (I and J). Scale bar = 50 μm. Abbreviations: p75-KO, p75NTR-knockout.


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Figure 5 proNGF changes the lineage fate of NSCs. (A) After being differentiated for seven days in the presence of 2 nM proNGF, NSCs showed a slight increase in the percentages of GFAP-positive astrocytes (a, green) and a significant decrease in CNPase- (b, red) and NG2(b, green) positive oligodendrocytes, but no changes in β-tubulin III-positive neurons (a, red). Nuclei were counterstained with Hoechst (a and b, blue). (B) Bar graphs showed the percentages of immuno-positive cells. (C) Immunoblotting showed the effects of proNGF on protein levels of GFAP, β-tubulin III, CNPase and NG2 in NSCs after a 7-day differentiation. β-Actin was used as a loading control. Real-time PCR was performed to determine the relative transcript levels of lineage-restricted genes in NSCs after the differentiation for 24 h (D) and 4 days (E). Values in bar graphs represent the mean ± SEM from three independent experiments. *p b 0.05, **p b 0.01, ***p b 0.001 between groups. Scale bar = 50 μm.

proNGF inhibited oligodendrogenesis in a p75NTR-dependent manner

that p75NTR is essential for the effect of proNGF on oligodendrocyte differentiation.

The above results demonstrated that proNGF caused a prominent change in the proportion of oligodendrocyte lineage. To determine whether p75NTR mediated the effect of proNGF on the differentiation of NSCs to oligodendrocyte, NSCs were treated with both p75NTR/Fc and 2 nM proNGF. p75NTR/Fc (140 nM) largely recovered the reduction of NG2-positive cells caused by proNGF (p b 0.05, vs. proNGF group, Figs. 6A and B). However, such an effect was not observed in CNPase-positive cells (p N 0.05, vs. proNGF group, Figs. 6A and C). p75NTR deficiency depleted the response of NSCs to proNGF under the differentiation conditions. Interestingly, p75NTR deficiency per se resulted in a significant decrease in both immature and mature oligodendrocytes (p b 0.001, vs. PBS group of wild-type cells, Figs. 6D–F). These observations support the notion

proNGF inhibited ERK 1/2 phosphorylation through p75NTR upon differentiation It has been reported that ERK1/2 phosphorylation is involved in growth factor-mediated oligodendrocyte lineage differentiation and increases Olig2 expression at early stages of oligodendrogenesis (Hu et al., 2008). Thus, the effect of proNGF on the phosphorylation of ERK1/2 under the differentiation conditions was investigated. After being cultured in DMEM/F12 medium without B27 and FBS supplements for 1 h, the differentiation of NSCs was induced by replacing the culture medium with DMEM/F12 containing B27 and 1% FBS, which led to a remarkable increase of ERK 1/2 phosphorylation levels (Fig. 7A). proNGF transiently inhibited ERK1/2 phosphorylation at 10 min (p b 0.05, vs. PBS group, Figs. 7B and C)


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Figure 6 proNGF inhibits oligodendrogenesis in a p75NTR-dependent manner. (A) Double immunostaining of NG2 (green) and CNPase (red) showed a restoration of oligodendrogenesis in proNGF-treated NSCs in the presence of p75NTR/Fc (140 nM), which was added simultaneously with proNGF to block its binding to p75NTR. Nuclei were stained with Hoechst (blue). Statistical analysis was shown in (B) and (C). (D) proNGF did not impact the percentages of NG2 (green) - and CNPase (red) -positive cells in p75NTR-knockout NSCs. Nuclei were stained with Hoechst (blue). Statistical analysis was shown in (E) and (F). *p b 0.05, **p b 0.01, ***p b 0.001 between groups. Values in bar graphs represent the mean ± SEM from three independent experiments. Scale bar = 50 μm. Abbreviations: WT, wild type; p75-KO, p75NTR-knockout; p75/Fc, p75NTR/Fc.

while the addition of p75NTR/Fc resulted in some restoration of ERK 1/2 activation (p b 0.01, vs. proNGF group, Figs. 7D and E). The phosphorylation of ERK 1/2 in p75NTR-knockout NSCs was also elevated upon differentiation, but was not affected by proNGF treatment (Figs. 7F–H), indicating that the inhibition of proNGF on ERK 1/2 phosphorylation is dependent on p75NTR signaling. To investigate whether ERK 1/2 played an essential role in oligodendrogenesis, NSCs were differentiated in the presence of U0126 for seven days. As shown in Figs. 7I and J, application of U0126 significantly reduced the proportions of NG2- and CNPase-positive cells, and increased the proportion of GFAPpositive cells compared with DMSO solvent control. However, the proportions of β-tubulin III-positive cells were not affected. A positive relationship between the inhibitory effect of U0126 on oligodendrogenesis and its concentration was observed. Notably, the phosphorylation level of ERK 1/2 was dramatically decreased by the presence of U0126 (10 μM) in the differentiation medium (Fig. 7K). Collectively, these data suggest that the inhibition of ERK 1/2 phosphorylation possibly contributed to the effect of proNGF on oligodendrogenesis.

Discussion In the present study, our data provide the first evidence of the critical role of proNGF in regulating the proliferation and differentiation of hippocampal NSCs from postnatal mice. To evaluate the effects of proNGF on NSC proliferation, we performed cell viability assay, BrdU incorporation assay and neurosphere assay. Our results showed that proNGF significantly reduced the total number of viable cells and the proportion of BrdU-positive cells, as well as the neurosphere size, suggesting that proNGF exerted an inhibitory role on NSC proliferation. Proliferation is intimately controlled by a progression in the cell cycle. Mitogenic control of proliferation occurs early in G1, allowing cells to bypass the first decision point (R1) in this cycle when a cell may either continue to proliferate or exit the cell cycle (Lange et al., 2009). The results of flow cytometry demonstrate that proNGF increases the distribution of cells in the G1/G0 phase at the expense of the S phase and downregulates the expression of cyclin E, an important positive regulator driving cells to pass the G1 phase. These findings are in accordance with the inhibitory effect of proNGF on NSC proliferation.

884 Our data showed that proNGF reduced the proportion of oligodendrocyte lineage after NSCs differentiated, accompanied by a slight increase in the proportion of astrocytes. However, the ratio of neurons was not substantially changed.

J. Guo et al. Previous reports have shown that transcription factors regulate cell-type-specific differentiation during CNS development. Olig1 and Olig2, two basic helix–loop–helix (bHLH) transcription factors, are believed to facilitate oligodendrogenesis

Effects of proNGF on hippocampal NSCs via p75NTR during CNS development (Rowitch et al., 2002). Moreover, downregulation of Olig2 has been shown to cause a switch from an oligodendrocyte to an astrocyte fate in neural stem cells (Zhu et al., 2012). In this study, Olig2 and Olig1 were found to be downregulated under the differentiation conditions after 24-hour and 4-day treatments of proNGF respectively, indicating that proNGF might inhibit oligodendrogenesis partially through regulating the expression of oligodendrocyte lineagespecific transcription factors. p75NTR, initially recognized as a low-affinity pan-receptor for the neurotrophin family, has been demonstrated to preferentially bind proNGF with the highest specificity and affinity (Lee et al., 2001). Recently, several studies have provided evidence for the involvement of p75NTR in NSC proliferation and differentiation. It was proposed that p75NTR defined a discrete population of highly proliferative SVZ precursor cells and that the absence of p75NTR disrupted neurotrophin-regulated neurogenesis in vivo (Young et al., 2007). p75NTR deficiency also significantly decreased the number of BrdU-positive cells in the hippocampus of adult mice (Bernabeu and Longo, 2010). In addition, p75NTR is essential for the action of BDNF and NT3 on oligodendrocyte differentiation (Du et al., 2006). In this study, we confirmed the expression of p75NTR on hippocampal NSCs in culture. The addition of p75NTR/Fc fusion protein into the culture medium as a scavenger of proNGF significantly reversed its effects on NSC proliferation and oligodendrocyte differentiation. Moreover, deletion of the ligand-binding extracellular domain of p75NTR abolished the responsiveness of NSCs to proNGF. These results demonstrated an important role of proNGF/p75NTR interaction in the functions of proNGF. It is interesting to note that p75NTR deficiency resulted in a remarkable decrease in NSC proliferation and oligodendrogenesis, which is consistent with previous studies (Bernabeu and Longo, 2010; Du et al., 2006). It is likely that p75NTR deficiency depletes the beneficial effects of certain ligands such as NGF and NT3 secreted by NSCs themselves, which has been shown to promote NSC proliferation and oligodendrogenesis. Another possibility is that p75NTR functions in a ligand-independent manner which might be inhibited by proNGF binding as proposed by Bredesen and Rabizadeh (1997). However, neither p75NTR deficiency (data not shown) nor proNGF treatment exerted an obvious effect on neurogenesis, which seems to be contradictory to

885 previous observations on SVZ NSCs (Young et al., 2007). It might be partially explained by a relatively low level of neuron differentiation in our experimental condition. Alternatively, NSCs from different regions of the brain are not all equivalent and have specific responses to stimulators (Thiriet et al., 2011). Furthermore, the downstream signaling pathways of p75NTR are so complicated that proNGF may induce quite different responses from those induced by mature neurotrophins. Although p75NTR has been correlated to the proliferation and differentiation of NSCs, the underlying molecular mechanisms remain undefined. ERK 1/2 activation has been shown to be responsible for the proliferation and oligodendrogenesis of NSCs, and to be able to promote cell cycle progression and Olig2 expression (Wang et al., 2009; Hu et al., 2008), which led us to investigate whether or not ERK 1/2 signaling pathway is regulated by proNGF/p75NTR interaction in NSCs. Using western blotting techniques, we observed that quiescent NSCs showed an increased phosphorylation level of ERK 1/2 upon the induction of proliferation and differentiation. Intriguingly, proNGF transiently inhibited the phosphorylation of ERK 1/2, which was demonstrated to be mediated by p75NTR. Both the proliferation of NSCs and their differentiation to oligodendrocyte were strongly inhibited when ERK 1/2 phosphorylation was impaired by U0126, suggesting that the ERK 1/2 signaling pathway played a critical role in the proliferation and lineage fate determination of NSCs. These results raised the possibility that the inhibition of ERK 1/2 signal by proNGF partially accounted for its negative regulation of NSC proliferation and oligodendrogenesis. It has been shown that proNGF prevented ERK 1/2 activation induced by BDNF in basal forebrain cholinergic neurons (Volosin et al., 2006). Furthermore, downregulation of p75NTR in mesencephalic dopaminergic neurons caused a prominent increase in the ERK 1/2 phosphorylation level, while a high expression level of p75NTR was accompanied by ERK 1/2 inhibition, indicating that ERK 1/2 phosphorylation might be inhibited by p75NTR (Alavian et al., 2009). However, Masoudi et al. showed that proNGF is capable of activating TrkA/ERK 1/2 in primed PC12 cells which highly express TrkA although p75NTR is also present (Masoudi et al., 2009). Considering the fact that proNGF exerts various effects depending on the presence of co-receptors of p75NTR and their balance, we performed western blot to detect the expression of sortilin and TrkA on

Figure 7 proNGF inhibits ERK 1/2 phosphorylation through p75NTR under the differentiation conditions. (A) Immunoblotting showed that the phosphorylation level of ERK1/2 in NSCs varied with time after differentiation. (B) proNGF transiently inhibited ERK1/2 phosphorylation in NSCs at 10 min after differentiation, as revealed by densitometric analysis in (C). (D) NSCs were treated with proNGF (2 nM) alone and together with p75NTR/Fc (140 nM) and ERK 1/2 phosphorylation level was determined. (E) The densitometric analysis showed that ERK 1/2 phosphorylation could be partially recovered by a combined treatment with p75NTR/Fc. (F) Immunoblotting showed a similar phosphorylation of ERK1/2 in p75NTR-knockout NSCs after differentiation. (G) proNGF did not impact ERK 1/2 phosphorylation in p75NTR-knockout NSCs at the indicated time points after differentiation, as shown by the densitometric analysis in (H). (I) Effects of U0126 at various concentrations on NSC differentiation were demonstrated by immunostaining with antibodies against GFAP (green, upper panel), β-tubulin III (red, upper panel), CNPase (red, lower panel) and NG2 (green, lower panel). Cells were counterstained with Hoechst (blue). (J) Quantification of immuno-positive cells showed that U0126 treatment caused a significant increase in the percentage of GFAP-positive astrocytes and a reduction in NG2- and CNPase-positive oligodendrocytes. No impacts were found in the number of β-tubulin III-positive neurons. (K) Immunoblotting showed that U0126 (10 μM) dramatically inhibited ERK 1/2 phosphorylation in NSCs at the indicated time points after the initiation of cell differentiation. Data were shown as a mean ± SEM of three independent experiments. *p b 0.05, **p b 0.01, ***p b 0.001 between groups (C and E) or compared with 0 μM U0126 group (K). &&& and ### (K), p b 0.001 compared with each corresponding 0 μM U0126 group. Scale bar = 50 μm. Abbreviations: p75-KO, p75NTR-knockout.

886 NSCs. As shown in Fig. S4, both receptors were expressed by NSCs, with a relatively low expression level of TrkA compared to neonate mouse brain lysate. Therefore, it is likely that the inhibitory effect of proNGF on ERK 1/2 activation in NSCs can be attributed to either a high p75NTR/TrkA expression ratio, the presence of certain as-yet-unidentified p75NTR coreceptors or particular intracellular signaling molecules, which remains to be further investigated. Many studies have shown that neurogenesis is impaired in the hippocampus of aging and AD brains, resulting in memory loss and cognition deficit, although controversy still exists (Lazarov and Marr, 2010; Rodriguez et al., 2008). In addition, white matter damage due to oligodendrocyte degeneration and insufficient oligodendrogenesis was considered to account for the pathology of some CNS diseases including AD, spinal cord injury, and multiple sclerosis (Desai et al., 2010; Yune et al., 2007; Keough and Yong, 2012). Increased expression of proNGF and p75NTR has been shown to induce cell death under these pathological conditions (Ibanez and Simi, 2012). A recent study has shown that non-peptide, small molecules that blocked proNGF/p75NTR interaction has promoted functional recovery after spinal cord injury (Tep et al., 2013). Therefore, the pharmacological intervention on proNGF/p75NTR signaling might be a promising approach not only in promoting neural cell survival, but also in regulating NSC proliferation and facilitating the myelination process, and thus promoting desired outcomes. In the end, due to the complexity of the in vivo conditions, the effects of proNGF on NSC proliferation and differentiation should be scrutinized on the basis of in vivo models so as to provide more valuable information for the potential of blocking proNGF/p75NTR interaction in the treatment of CNS disorders. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scr.2013.05.004.

Disclosure of potential conflicts of interests The authors indicate no potential conflicts of interest.

Acknowledgment We gratefully acknowledge the support from the National Natural Science Foundation of China (81070967; 81271338), the Natural Science Foundation of Jiang Su Province (BK2006150; BK2009296), a program granted for scientific innovation research of college graduate in Jangsu Province (CXLX11_0803), the Fundamental Research Funds for the Central Universities (JKY2011016) and the Initial Fund of China Pharmaceutical University (to H.L.). We wish to thank Professor Moses Chao from New York University for providing the p75NTR antibody. We also wish to thank Ms Kate Rees from the University of South Australia and Mr Abdelkader Daoud from China Pharmaceutical University for reading and improving the manuscript.

References Alavian, K.N., Sgado, P., Alberi, L., et al., 2009. Elevated P75NTR expression causes death of engrailed-deficient midbrain dopaminergic neurons by Erk1/2 suppression. Neural Dev. 4, 11. Al-Shawi, R., Hafner, A., Olsen, J., et al., 2008. Neurotoxic and neurotrophic roles of proNGF and the receptor sortilin in the adult and ageing nervous system. Eur. J. Neurosci. 27, 2103–2114.

J. Guo et al. Beattie, M.S., Harrington, A.W., Lee, R., et al., 2002. ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury. Neuron 36, 375–386. Bernabeu, R.O., Longo, F.M., 2010. The p75 neurotrophin receptor is expressed by adult mouse dentate progenitor cells and regulates neuronal and non-neuronal cell genesis. BMC Neurosci. 11, 136. Bredesen, D.E., Rabizadeh, S., 1997. p75NTR and apoptosis: Trkdependent and Trk-independent effects. Trends Neurosci. 20, 287–290. Cattaneo, E., McKay, R., 1990. Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor. Nature 347, 762–765. Chakravarthy, B., Menard, M., Ito, S., et al., 2012. Hippocampal membrane-associated p75NTR levels are increased in Alzheimer's disease. J. Alzheimers Dis. 30, 675–684. Cragnolini, A.B., Friedman, W.J., 2008. The function of p75NTR in glia. Trends Neurosci. 31, 99–104. Darzynkiewicz, Z., Juan, G., 2001. DNA content measurement for DNA ploidy and cell cycle analysis. Current Protocols in Cytometry/ Editorial Board, J. Paul Robinson, Managing Editor … [et al.] (Chapter 7, Unit 7 5). Desai, M.K., Mastrangelo, M.A., Ryan, D.A., et al., 2010. Early oligodendrocyte/myelin pathology in Alzheimer's disease mice constitutes a novel therapeutic target. Am. J. Pathol. 177, 1422–1435. Du, Y., Fischer, T.Z., Clinton-Luke, P., et al., 2006. Distinct effects of p75 in mediating actions of neurotrophins on basal forebrain oligodendrocytes. Mol. Cell. Neurosci. 31, 366–375. Fahnestock, M., Michalski, B., Xu, B., et al., 2001. The precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in Alzheimer's disease. Mol. Cell. Neurosci. 18, 210–220. Hempstead, B.L., 2002. The many faces of p75NTR. Curr. Opin. Neurobiol. 12, 260–267. Hempstead, B.L., 2009. Commentary: regulating proNGF action: multiple targets for therapeutic intervention. Neurotox. Res. 16, 255–260. Hu, J.G., Fu, S.L., Wang, Y.X., et al., 2008. Platelet-derived growth factor-AA mediates oligodendrocyte lineage differentiation through activation of extracellular signal-regulated kinase signaling pathway. Neuroscience 151, 138–147. Ibanez, C.F., Simi, A., 2012. p75 neurotrophin receptor signaling in nervous system injury and degeneration: paradox and opportunity. Trends Neurosci. 35, 431–440. Kang, M.K., Kang, S.K., 2008. Interleukin-6 induces proliferation in adult spinal cord-derived neural progenitors via the JAK2/STAT3 pathway with EGF-induced MAPK phosphorylation. Cell Prolif. 41, 377–392. Keough, M.B., Yong, V.W., 2012. Remyelination therapy for multiple sclerosis. Neurotherapeutics. J. Am. Soc. Exp. Neurother. 10, 44–54. Lange, C., Huttner, W.B., Calegari, F., 2009. Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors. Cell Stem Cell 5, 320–331. Lazarov, O., Marr, R.A., 2010. Neurogenesis and Alzheimer's disease: at the crossroads. Exp. Neurol. 223, 267–281. Lee, R., Kermani, P., Teng, K.K., et al., 2001. Regulation of cell survival by secreted proneurotrophins. Science (New York, N.Y.) 294, 1945–1948. Liao, H., Huang, W., Schachner, M., et al., 2008. Beta 1 integrinmediated effects of tenascin-R domains EGFL and FN6-8 on neural stem/progenitor cell proliferation and differentiation in vitro. J. Biol. Chem. 283, 27927–27936. Masoudi, R., Ioannou, M.S., Coughlin, M.D., et al., 2009. Biological activity of nerve growth factor precursor is dependent upon relative levels of its receptors. J. Biol. Chem. 284, 18424– 18433.

Effects of proNGF on hippocampal NSCs via p75NTR Migita, H., Kominami, K., Higashida, M., et al., 2008. Activation of adenosine A1 receptor-induced neural stem cell proliferation via MEK/ERK and Akt signaling pathways. J. Neurosci. Res. 86, 2820–2828. Nunez, R., 2001. DNA measurement and cell cycle analysis by flow cytometry. Curr. Issues Mol. Biol. 3, 67–70. Pedraza, C.E., Podlesniy, P., Vidal, N., et al., 2005. Pro-NGF isolated from the human brain affected by Alzheimer's disease induces neuronal apoptosis mediated by p75NTR. Am. J. Pathol. 166, 533–543. Podlesniy, P., Kichev, A., Pedraza, C., et al., 2006. Pro-NGF from Alzheimer's disease and normal human brain displays distinctive abilities to induce processing and nuclear translocation of intracellular domain of p75NTR and apoptosis. Am. J. Pathol. 169, 119–131. Rodriguez, J.J., Jones, V.C., Tabuchi, M., et al., 2008. Impaired adult neurogenesis in the dentate gyrus of a triple transgenic mouse model of Alzheimer's disease. PLoS One 3, e2935. Rowitch, D.H., Lu, Q.R., Kessaris, N., et al., 2002. An ‘oligarchy’ rules neural development. Trends Neurosci. 25, 417–422. Shetty, A.K., Turner, D.A., 1998. In vitro survival and differentiation of neurons derived from epidermal growth factor-responsive postnatal hippocampal stem cells: inducing effects of brainderived neurotrophic factor. J. Neurobiol. 35, 395–425. Tep, C., Lim, T.H., Ko, P.O., et al., 2013. Oral administration of a small molecule targeted to block proNGF binding to p75 promotes myelin sparing and functional recovery after spinal cord injury. J. Neurosci. 33, 397–410. Terry Jr., A.V., Kutiyanawalla, A., Pillai, A., 2011. Age-dependent alterations in nerve growth factor (NGF)-related proteins,

887 sortilin, and learning and memory in rats. Physiol. Behav. 102, 149–157. Thiriet, N., Agasse, F., Nicoleau, C., et al., 2011. NPY promotes chemokinesis and neurogenesis in the rat subventricular zone. J. Neurochem. 116, 1018–1027. Volosin, M., Song, W., Almeida, R.D., et al., 2006. Interaction of survival and death signaling in basal forebrain neurons: roles of neurotrophins and proneurotrophins. J. Neurosci. 26, 7756–7766. Volosin, M., Trotter, C., Cragnolini, A., et al., 2008. Induction of proneurotrophins and activation of p75NTR-mediated apoptosis via neurotrophin receptor-interacting factor in hippocampal neurons after seizures. J. Neurosci. 28, 9870–9879. Wang, B., Gao, Y., Xiao, Z., et al., 2009. Erk1/2 promotes proliferation and inhibits neuronal differentiation of neural stem cells. Neurosci. Lett. 461, 252–257. Wang, Y.J., Valadares, D., Sun, Y., et al., 2010. Effects of proNGF on neuronal viability, neurite growth and amyloid-beta metabolism. Neurotox. Res. 17, 257–267. Wurmser, A.E., Palmer, T.D., Gage, F.H., 2004. Neuroscience. Cellular interactions in the stem cell niche. Science (New York, N.Y.) 304, 1253–1255. Young, K.M., Merson, T.D., Sotthibundhu, A., et al., 2007. p75 neurotrophin receptor expression defines a population of BDNFresponsive neurogenic precursor cells. J. Neurosci. 27, 5146–5155. Yune, T.Y., Lee, J.Y., Jung, G.Y., et al., 2007. Minocycline alleviates death of oligodendrocytes by inhibiting pro-nerve growth factor production in microglia after spinal cord injury. J. Neurosci. 27, 7751–7761. Zhu, X., Zuo, H., Maher, B.J., et al., 2012. Olig2-dependent developmental fate switch of NG2 cells. Development.